Phosphorylation of ciliary target proteins

描述（由申请方提供）：在气道疾病急性加重中，气道酸化，通过哮喘、COPD和支气管扩张的呼出气冷凝液中的低pH值测量。临床上，β-肾上腺素能激动剂用于通过刺激跨膜腺苷酸环化酶（tmAC）来增加cAMP，目的是在疾病恶化期间增强粘膜纤毛转运和支气管扩张。对于支气管扩张，吸入药物需要穿过气道上皮。目前使用的大多数支气管扩张剂在生理或酸性pH下具有正电荷。因此，这些药物不能自由扩散穿过气道上皮以到达其靶点。我们已经表明，pH依赖性有机阳离子/肉毒碱转运蛋白OCTN 1和OCTN 2在气道上皮细胞中表达，并且它们以pH依赖性方式将阳离子（包括沙丁胺醇）转运到气道上皮细胞中。在酸性气道环境中，带正电荷的支气管扩张剂的摄取减少，表明其支气管扩张作用将降低。我们还表明，细胞内酸化直接抑制纤毛跳动。作为一种对抗措施，增加细胞内HCO 3刺激纤毛通过一个途径，涉及cAMP生产的一种新的，胞质（或可溶性）腺苷酸环化酶（sAC）。这些新的发现表明pHi和HCO 3-在调节人体气道内稳态中具有重要作用。我们推测，pH直接和HCO 3-，可能通过sAC，在调节气道上皮细胞功能，包括摄取带电阳离子，如支气管扩张剂，发挥关键作用。这一假设将通过三个具体目标进行全面测试，包括评估吸入沙丁胺醇扩张正常人类志愿者气道血管能力的生物测定。目的1将检验cAMP产生由tmAC和sAC两者完成并且因此受HCO 3-、pH、Ca 2+的变化影响的假设。目的2将检验以下假设：气道上皮细胞的pH依赖性阳离子摄取将使在酸化期间气道表面上的μ-肾上腺素能激动剂以更高浓度可用。目的3将检验气道酸化抑制人气道粘膜下组织经上皮吸收阳离子（包括沙丁胺醇）的假设。因此，本申请使用翻译和最先进的方法解决了上皮表面上以及上皮下组织中的粘膜纤毛清除和药物可用性的新的和临床相关的调节机制。公共卫生相关性。气道疾病，特别是其恶化在美国和世界范围内构成重要的健康问题。在疾病加重期间气道酸化，预计pH值的这种变化会减少支气管扩张剂穿过气道上皮到达其靶点（即平滑肌）的摄取，并对粘膜纤毛清除率产生不利影响。本申请研究了支气管扩张剂进入气道粘膜的机制和粘膜纤毛清除调节的新方面。改善支气管扩张剂在疾病加重期间达到其目标的方式并预防粘膜纤毛功能障碍或将清除率恢复至正常水平是最终的临床目标。因此，该建议可以确定新的治疗靶点，有助于减轻气道疾病加重对个人和社会的负担。

DESCRIPTION (provided by applicant): In airway disease exacerbations, the airway acidifies as measured by low pH in exhaled breath condensate in asthma, COPD and bronchiectasis. Clinically, ¿-adrenergic agonists are used to increase cAMP via stimulation of transmembrane adenylyl cyclases (tmACs), with the intent to enhance mucociliary transport and bronchodilation during disease exacerbations. For bronchodilation, inhaled drugs need to cross the airway epithelium. The majority of the currently used bronchodilators have a positive charge at physiological or acidic pH. Thus, these drugs cannot freely diffuse across the airway epithelium to reach their targets. We have shown that the pH-dependent organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in airway epithelia and that they transport cations, including albuterol, into airway epithelial cells in a pH-dependent manner. Uptake of positively charged bronchodilators is decreased in an acidic airway environment, suggesting that their bronchodilatory effects will be reduced. We have also shown that intracellular acidification directly inhibits ciliary beating. As a counter measure, increases in intracellular HCO3- stimulate cilia via a pathway involving cAMP production by a novel, cytosolic (or soluble) adenylyl cyclase (sAC). These new findings suggest a significant role for pHi and HCO3- in the regulation of human airway homeostasis. We hypothesize that pH directly and HCO3-, likely through sAC, play critical roles in regulating airway epithelial cell functions, including uptake of charged cations such as bronchodilators. This hypothesis will be comprehensively tested with three specific aims, including a bioassay that assesses the ability of inhaled albuterol to dilate airway blood vessels in normal human volunteers. Aim 1 will test the hypothesis that cAMP production is accomplished by both tmACs and sAC and thus influenced by changes in HCO3-, pH, Ca2+. Aim 2 will test the hypothesis that pH-dependent cation uptake into airway epithelial cells will make ¿-adrenergic agonists available in higher concentrations on the airway surface during acidification. Aim 3 will test the hypothesis that airway acidification inhibits transepithelial cation uptake, including albuterol, into airway submucosal tissues in human beings. This application thus addresses a novel and clinically relevant regulation mechanism of mucociliary clearance and of drug availability on the epithelial surface as well as in the subepithelial tissue using a translational and state-of-the-art approach. PUBLIC HEALTH RELEVANCE. Airway diseases and specifically their exacerbations pose important health problems in the USA and worldwide. The airway acidifies during disease exacerbations and this change in pH is predicted to decrease uptake of bronchodilators across the airway epithelium to their target (namely smooth muscle) and adversely affect aspects of mucociliary clearance. This application examines mechanisms by which bronchodilators are taken up into the airway mucosa and novel aspects of mucociliary clearance regulation. Improving ways that bronchodilators reach their target during disease exacerbations and preventing mucociliary dysfunction or restoring clearance to normal levels is the ultimate clinical goal. This proposal thus may identify new therapeutic targets that could help to decrease the burden from airway disease exacerbations on individuals and on society.